Limited Neutralization of Omicron by Antibodies from the BNT162b2 Vaccination against SARS-CoV-2

Since early December 2021, the omicron variant has posed additional challenges to the world-wide management of the SARS-CoV-2 pandemic. Immune evasion is a key factor for its increased transmissibility. While serological studies have measured levels of neutralizing antibodies in response to vaccines, our understanding of the humoral immune response to omicron on a single-antibody level is limited. Here, we characterize a set of BNT162b2 vaccine-derived antibodies for neutralization of omicron pseudovirus. We show that approximately 50% of neutralizing anti-RBD antibodies cross-neutralize omicron, albeit with lower potency than the original Wuhan-Hu1 strain. All investigated neutralizing anti-S2 antibodies cross-neutralize omicron, however all of them are less potent than anti-RBD antibodies. While additional booster immunizations of the current vaccine generate increased antibody levels and better protection, we anticipate that the second generation of vaccines will yield more high-affinity antibodies against omicron.


Introduction
Within less than two months, the B.1.1.529 variant (omicron) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), replaced B.1.617.2 (delta) as the most dominant strain worldwide. With 37 amino acid substitutions in the Spike protein (S), it marks a major antigenic shift from the original Wuhan-Hu1 and the delta sequence. Immune evasion is a major contributing factor for omicron's improved transmissibility [1][2][3] . To date, all approved vaccines against SARS-CoV-2 are based on the Wuhan-Hu1 sequence of the S protein. Serologic studies have shown that convalescent patient plasma and plasma from individuals vaccinated with BNT162b2 neutralize omicron 44-fold and 12-fold less effectively than delta 2 . A third immunization with BNT162b2 (booster) increases neutralization e cacy by 10 to 100-fold 2,4 . Accordingly, the booster prevents 88% of omicronrelated hospitalizations, as opposed to only 52% at 25 + weeks post second vaccination 5,6 . This increased e cacy should be attributed to higher anti-S antibodies with increased a nity to Wuhan-Hu1 -and to omicron only by proxy. While sera and plasma neutralization levels have been established, the anti-omicron response to BNT162b2 is not well understood on a single antibody level.
We recently investigated the acute B cell response to the BNT162b2 vaccine on a single-cell level and discerned the development of anti-receptor binding domain (RBD) and anti-S2 antibodies from naïve and memory B cells, respectively 7 . The RBD is located on the S1 domain of the S protein and interacts with human angiotensin-converting enzyme 2 (ACE2) to initiate viral cell entry, whereas the S2 domain facilitates viral cell membrane fusion 8, 9 . Most neutralizing antibodies found in COVID-19 patients target RBD 10 . Neutralizing antibodies against the S2 subunit have been described, but they generally neutralize SARS-CoV-2 less e ciently than anti-RBD mAbs 11,12 . However, we have shown that anti-S2 mAbs develop early in response to vaccination, and are cross-reactive to other betacoronaviruses due to higher structural conservation of the S2 subunit over S1 7,13,14 . Anti-S2 antibodies could therefore be crucial for the protection against novel variants. We previously expressed and tested 50 vaccine-derived monoclonal antibodies (mAbs) against RBD/S1 and S2, and identi ed 15 anti-RBD mAbs that neutralized Wuhan-Hu1 and delta 7 .
Here, we added additional 55 mAbs from the same sequence dataset and investigated their binding to RBD and S2 as well as their neutralization potency to Wuhan-Hu1, delta, and omicron.

Discussion
We investigated vaccine-derived antibody neutralization of omicron on a single-antibody level and showed that approximately 50% of anti-RBD antibodies neutralize omicron, albeit with a 19-fold lower potency. Strikingly, the most potent neutralizing antibodies against omicron had a 46-fold decreased potency compared to the best neutralizing antibodies against Wuhan-Hu1. As highly potent neutralizing antibodies are important for immune protection, this difference is substantial, and while additional booster immunizations with the original BNT162b2 vaccine will increase antibody levels, it is unlikely to generate larger amounts of higha nity anti-omicron-RBD antibodies.
For anti-S2 mAbs, the rate of cross-neutralization against omicron is higher (7 out of 7 mAbs in our study). Anti-S2 antibodies stem from a recall response of memory B cells against prior infections with heterologous betacoronaviruses 7,13,14 . Their reactivity is broader and less susceptible to immune escape by novel variants. The Evolution of omicron has likely been driven by anti-RBD antibodies rather than anti-S2 antibodies (mutation rate RBD: 7.77/100 amino acids, S2: 1.02/100 amino acids). However, overall neutralization potency is 16.9-fold lower than for anti-RBD antibodies, and anti-S2 antibodies therefore likely contribute limited protection. Vaccine strategies that generate broadly-neutralizing anti-S2 antibodies and therapeutic strategies with high-potency anti-S2 monoclonal antibodies 14 have promise to work effectively against novel variants.
In conclusion, our neutralization data on vaccine-derived monoclonal antibodies is in line with serological studies, as we show antibodies with decreased neutralization potency against omicron. Vaccine updates will likely achieve high-potency anti-omicron antibody responses.

Materials And Methods
Recombinant expression and puri cation of monoclonal antibodies (mAbs). All heavy and light-chain (HC and LC) sequences were obtained by single-cell repertoire sequencing from individuals on days 7, 21, and 28 post initial BNT162b2 vaccination (second immunization on day 21) 7 . Codon-optimized HC and LC variable sequences were cloned into in-house vectors, containing human IgG1 and κ or λ constant regions, respectively. HC and LC plasmids were transfected into Expi293F cells using FectoPro (Polyplus transfection). Cell supernatants were collected after 7 days and puri ed with AmMag Protein A beads (Genscript). mAb concentrations were measured using a nanodrop spectrophotometer (Thermo Fisher Scienti c) and human IgG quantitation ELISAs (Bethyl Laboratories). which was then ampli ed in E.Coli and checked for correct insertion and sequence integrity by Sanger-sequencing. Pseudotyped lentiviral particles were generated as previously described 7,15 . Brie y, LentiX 293T cells in 10-cm tissue culture dishes were transfected 24 h post-seeding using Fugene transfection reagent (Promega) with pHAGE-CMV-Luc2-IRES-ZsGreen-W, lentiviral packaging plasmids (HDM-Hgpm2, HDM-tat1b and pRC-CMV-Rev1b) and wild-type or variant SARS-CoV-2 spike plasmids. 48-60 h post transfection, viral supernatants were collected and spun at 500g for 10 min. Lentiviral supernatants were concentrated with LentiX concentrator (Takara) according to the manufacturer's instructions. Pellets were resuspended in EMEM at approximately 1/100 of the initial amount of media, and stored at − 80°C until use. Virus was titrated on HeLa-ACE2 cells, provided by Dennis Burton at the Scripps Research Institute.
Neutralization assays were performed as previously described 7,15 . Brie y, HeLa-ACE2 cells were seeded at 12,500 cells/well in atbottom 96-well plates 20h before viral transduction. mAbs were prepared in EMEM in eight ve-fold serial dilutions starting at 50 or 10 µg ml − 1 , incubated with SARS-CoV-2 pseudotyped virus for 1h at RT, and then added to HeLa-ACE2 cells in the presence of 5 µg ml − 1 polybrene (Sigma Millipore). After 48h, luciferase activity was measured using the Britelite plus Reporter Gene Assay System (Perkin Elmer), and read on a GloMax Explorer Microplate Reader (Promega). Neutralization assays were performed 1-3 times in at least triplicates for each dilution. mAbs were considered neutralizing only if a considerable decrease in luminescence was measured at concentrations < 10 µg/ml ( Supplementary Figs. 1-2).
Human subjects. No human subjects were included in this study. Antibody sequences were derived from our prior study 7   values of anti-RBD mAbs against Wuhan-Hu1, delta, and omicron. Medians ± interquartile ranges are shown; ns, not signi cant, unpaired two-tailed Kruskal-Wallis test.

Supplementary Files
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